ABSTRACT The heart must adjust its contractile force to counteract the blood pressure changes (due to physical activity, body position, emotional stress, etc.) to maintain cardiac output. Cardiac muscle cells possess the intrinsic ability to sense mechanical load and fine-tune contractile force accordingly. Previous work by us and others found that increasing mechanical loading on muscle strips or single myocytes results in an increase of the cytosolic Ca2+ transient (CaT) that increases the contractile force to partially compensate for the added mechanical load. A sustained increase in CaT requires augmenting the Ca2+ content of the sarcoplasmic reticulum (SR), which must come from a net increase of Ca2+ across the sarcolemma. However, the mechanism that links mechanical loading to the control of Ca2+ entry remains unknown. Plausible mechanisms for this net increase include a mechanical load-induced increase of Ca2+ influx through L-type Ca2+ channels (ICaL) or a decreased efflux from Na+-Ca2+ exchanger (INCX). However, studying how mechanical loading affects ICaL or INCX has been hampered by the technical difficulty of subjecting myocytes to mechanical loads while simultaneously doing patch-clamp experiments. Now we have overcome this obstacle. Innovation: Recently we invented the Patch-Clamp-in-Gel technique that enables us to simultaneously measure membrane voltage or current, SR or cytosolic Ca2+, and contraction in single myocytes embedded in a viscoelastic hydrogel that controls mechanical load on the cell. Preliminary data reveal that mechanical load increases ICaL, prolongs the action potential (AP), and increases SR Ca2+ content in rabbit ventricular myocytes. Therefore we hypothesize that mechanical load increases Ca2+ entry during AP to elevate the SR Ca2+ content and Ca2+ release that increases CaT, which can partially compensate for increased mechanical load. While a compensatory increase in CaT might be beneficial to offset moderate increases in mechanical loading, we further posit that excessive loading causes SR Ca2+ overload by excessive ICaL increase or INCX decrease, leading to arrhythmogenic spontaneous Ca2+ release. Failing hearts cannot generate enough force to maintain adequate cardiac output. We hypothesize that the compensatory increase of CaT with increased load is blunted in HF. Our interdisciplinary team will develop the new Patch-Clamp-in-Gel technique and test the hypotheses using ventricular myocytes from healthy and HF rabbit, and transgenic mouse to achieving three specific aims. (1) Systematically investigate how mechanical load regulates myocyte E-C- coupling during cardiac cycle. (2) Decipher mechano-electro-transduction effects on ICaL and INCX and Ca2+ entry during AP. (3) Understand how pathological high load may cause Ca2+ dysregulation and arrhythmogenic activities in HF. The outcome of this project will elucidate how mechanical load affects cardiac excitation-contraction coupling in compensatory response to physiological loading, and how mechanotransduction is impaired in heart failure patients to cause arrhythmias and contractile dysfunction.